Received December 11, 2014; Revision received February 24, 2015
O-Antigens (O-specific polysaccharides) of Shigella flexneri, a
primary cause of shigellosis, are distinguished by a wide diversity of
chemical modifications following the oligosaccharide O-unit assembly.
The present review is devoted to structural, serological, and genetic
aspects of these modifications, including O-acetylation and
phosphorylation with phosphoethanolamine that have been identified
recently. The modifications confer the host with specific
immunodeterminants (O-factors or O-antigen epitopes), which accounts
for the antigenic diversity of S. flexneri considered as a
virulence factor of the pathogen. Totally, 30 O-antigen variants have
been recognized in these bacteria, the corresponding O-factors
characterized using specific antibodies, and a significant extension of
the serotyping scheme of S. flexneri on this basis is suggested.
Multiple genes responsible for the O-antigen modifications and the
resultant serotype conversions of S. flexneri have been
identified. The genetic mechanisms of the O-antigen diversification by
acquisition of mobile genetic elements, including prophages and
plasmids, followed occasionally by gene mobilization and inactivation
have been revealed. These findings further our understanding of the
genetics and antigenicity of S. flexneri and assist control of
shigellosis.KEY WORDS: Shigella flexneri, O-antigen, O-polysaccharide
structure, serotype-converting bacteriophage, transposon, plasmid,
serotyping, immunodeterminant

Shigellosis, or bacillary dysentery, is an acute diarrheal disease that
remains an important public health challenge, especially in developing
countries. There are an estimated about 164.7 million shigellosis cases
annually worldwide causing 1.1 million deaths, with the majority
involving children under five years old [1]. The
causative agent of shigellosis is Shigella spp., nonmotile,
nonspore-forming facultative anaerobic Gram-negative bacteria, which
are among the bacterial pathogens most frequently isolated from
patients with diarrhea. Invasion by these bacteria of the colonic and
rectal mucosa and the following inflammatory response provoke massive
mucosal destruction reflected in strong abdominal cramps and stools
containing blood and mucus [2]. Based on
biochemical properties and O-antigen specificity, the genus
Shigella is divided into four species or subgroups, including
S. dysenteriae, S. flexneri, S. boydii, and S.
sonnei [3], although genetically they all but
S. boydii type 13 are clones of Escherichia coli
[4]. Shigella flexneri is the predominant
species causing shigellosis in developing countries and the second,
after S. sonnei, most common in industrialized countries [5-7].

Being a serologically heterogeneous species, S. flexneri is
further divided into various serotypes and subtypes. A commercially
available monovalent antisera kit (Denka Seiken, Japan) and monoclonal
antibody reagents (MASFs) (Reagensia AB, Sweden) are widely used for
serotyping S. flexneri isolates. The serospecificity of S.
flexneri is defined by a combination of immunodeterminants
(O-factors), which reside on the O-antigen located on the bacterial
cell surface [3]. The O-antigen, also called
O-specific polysaccharide or O-polysaccharide, is a part of the
outer-membrane lipopolysaccharide (LPS) and is linked to the lipid
moiety (lipid A) via a core oligosaccharide. The O-polysaccharide
consists of many oligosaccharide repeats (O-units). Structure and
serology of the O-antigens of S. flexneri have been intensively
studied for the last 50 years ([8-10] and references therein).

The O-antigens of S. flexneri are synthesized by the O-antigen
polymerase (Wzy)/flippase (Wzx)-dependent pathway, whereby the O-unit
is preassembled on a lipid carrier on the cytoplasmic side of the inner
membrane, and after translocation (flipping) to the periplasmic side
mediated by Wzx, is polymerized by Wzy with participation of a chain
length regulator Wzz. There are two basal S. flexneri
O-polysaccharide structures and, correspondingly, two non-variable gene
clusters for O-antigen biosynthesis: one for serotype 6 and the other
for the remaining serotypes. The bacteria of the two groups have
different evolutionary origins and belong to different lineages of
Shigella clones of E. coli [4]. As in
most E. coli clones including the other Shigella spp.,
the O-antigen gene cluster maps between the housekeeping genes
galF and gnd on the chromosome [11].
It contains genes for synthesis of a nucleotide (deoxythymidine
diphosphate) precursor of l-rhamnose, a specific monosaccharide
component of both basal O-polysaccharides, for glycosyltransferases
necessary for the assembly of the O-unit, and O-antigen processing
genes: wzx for flippase and wzy for O-antigen
polymerase.

Molecular typing targeting specific genes in the O-antigen cluster,
including wzx, wzy, and glycosyltransferases genes,
detects all S. flexneri non-6 serotypes as a single group [12]. Escherichia coli O13, O129, and O135
having the same basal O-antigen structure [10, 13] and essentially identical O-antigen gene cluster
[11] also fall in this group, and, similarly,
E. coli O147 forms one molecular group and shares the O-antigen
structure with S. flexneri serotype 6 [11,
12]. The closely related E. coli and S.
flexneri clones can be differentiated using PCR assays based on
other genes not related to the O-antigen synthesis [12].

The O-antigens of S. flexneri non-6 serotypes are highly diverse
due to various chemical modifications to the basal structure giving
rise to the observed serological heterogeneity [3].
A number of genes outside the O-antigen cluster are involved in the
modifications [14], which occur after the O-unit
assembly and before the transfer of the mature O-polysaccharide to the
lipid A-core region of the LPS. A molecular approach targeting specific
O-antigen modification genes identified by that time has been developed
for serotyping S. flexneri within the group of non-6 serotypes
[15].

The O-antigen plays an important role in the pathogenesis of S.
flexneri; particularly, it protects the bacteria from the lytic
action of serum complement and promotes adherence and internalization
of bacteria to intestinal epithelial cells [16-18]. Creating antigenic diversity by O-antigen
modifications is considered as an important virulence factor of S.
flexneri that enhances survival of the pathogens because the host
has to mount a speciﬁc immune response to each serotype [19]. Moreover, such modification as glucosylation at
certain sites promotes invasion of S. flexneri into host cells
mediated by the type III secretion system [16].

In previous reviews devoted to S. flexneri O-antigens [9, 14, 19],
modifications known by that time, including glucosylation at various
sites and O-acetylation at one site (on RhaI), have been
considered in detail. Recently, more sites of O-acetylation [10, 13, 20-25] and a novel modification
type, phosphorylation with phosphoethanolamine (PEtN) [26-29], have been identified and
genetic bases of the new modifications have been elucidated. The
present review summarizes structural, serological, and genetic aspects
of the O-antigen modifications in S. flexneri with emphasis on
the new findings.

The polysaccharide (1) present in serotype Y is characterized by two
antigenic specificities labeled dual group O-factor 3,4. A structural
domain that defines this O-factor has not been completely identified
yet [31, 32]. In some cases,
its manifestation is ambiguous as strains otherwise identical in the
O-antigen structure and the presence of other immunodeterminants may
express or may not express O-factor 3,4 (e.g. former serotypes 3b and
3c, which have been proposed to be combined into one serotype 3b [10]). The polysaccharide (1) can be modified by
adding various chemical groups (α-d-glucopyranosyl, O-acetyl,
phosphoethanolamine) to different sugars giving rise to enormously
diverse O-antigen structures and, correspondingly, to serological
heterogeneity, which is the basis for serotyping of S. flexneri
strains (table).

Structures of the O-polysaccharides of S. flexneri. Included are
both serotypes already approved internationally and provisional
serotypes that express epitopes associated with newly identified
O-acetyl and PEtN groups

Note: RhaIII and GlcNAc are O-acetylated
non-stoichiometrically. A minor 4-O-acetylation on RhaIII
that occurs alternatively to the major 3-O-acetylation on
RhaIII is not shown. In the antigenic formulae, type and
group O-factors are indicated before and after colon, respectively.
O-factor 3,4 associated with the O-polysaccharide backbone is variably
expressed and, except for serotype Y, is omitted from the antigenic
formulae in the table.a-e Authors’ unpublished data.a Strain 2005122; the degree of 6-O-acetylation on GlcNAc
~75%.b Strain 2001019.c Strain 2005128; the degree of 6-O-acetylation on GlcNAc
~75%.d Strain 06AH74; the degree of 3/4-O-acetylation on
RhaIII ~40/25%.e Strains 06HN054 and 06HN303.f Proposed based on the reactivity with MASF IV-1 [45, 46] with no data on the
O-antigen structure and genetics available.

Glucosylation may occur on any monosaccharide in the polysaccharide (1)
giving rise to type O-factors I, II, IV, and V when present at
various positions on RhaI, RhaII, or GlcNAc, and
to dual group O-factor 7,8 when present on RhaIII
(Fig. 1). The type O-factors define serotypes
1, 2, 4, and 5, respectively, whereas the group O-factor 7,8 may be
expressed in different serotypes and occur in combination with various
type O-factors [8, 9, 34, 35] (table). As a result, in
the O-units of some subtypes there are two side-chain glucosyl groups.
The degree of glucosylation at each position is close to
stoichiometric, but the first O-unit of the O-polysaccharide chain
linked to the LPS core lacks any glucosyl residue [39-41].

In serotype 7, GlcNAc carries the
α-d-Glcp-(1→2)-α-d-Glcp-(1→
disaccharide [37, 38], which
defines O-factor VII (IC). Subtype 7a was originally called 1c [37], but wild-type strains of this subtype do not
react with antibodies against O-factor I. Therefore, it was suggested
to rename it 7a and to replace the type O-factor IC with VII in the
serotyping scheme of S. flexneri [38].

O-Acetylation has been identified on RhaI, RhaIII,
and GlcNAc [10, 22-24, 33, 38,
42] (Fig. 1). The degree
of O-acetylation is variable and depends likely not only on strain but
also on storage and cultivation conditions. On RhaI, the
2-O-acetylation is stoichiometric or close to stoichiometric, whereas
on GlcNAc, 6-O-acetylation varies from 30 to 75%. RhaIII is
O-acetylated at position 3 in some O-units and at position 4 in some
others (3/4-O-acetylation), the former being the major (25-70%) and the
latter the minor (15-25%) modification site. All combinations of
O-acetylated and non-acetylated RhaIII and GlcNAc have been
found in the O-units of the serotype 2a O-polysaccharide, and hence
O-acetylation on both residues is random [20]. In
a short-chain LPS having a single O-unit, GlcNAc lacks 6-O-acetylation
and RhaIII is mono-O-acetylated at any position [40].

O-Acetylation on RhaI, RhaIII, and GlcNAc defines
group O-factors 6, 9, and 10, respectively. Serotype 3 strains express
type O-factor III, which depends on the same 2-O-acetyl group on
RhaI as group O-factor 6 [42] but, in
contrast to the latter, is abolished by glucosylation on GlcNAc in
serotypes 1b, 4b, and 7b. O-Factor III could be recovered from
serotypes 1b and 4b by transformation with the functional oacD
gene, which resulted in partial 6-O-acetylation (~25-30%) accompanied
by deglucosylation on GlcNAc [24]. 6-O-Acetylation
on GlcNAc also occurs in the common enterobacterial polysaccharide
antigen [43, 44] and, as a
result, O-factor 10 is expressed by some other enteric bacteria,
including Shigella sonnei phase II [24].

In early studies of S. flexneri O-antigen structures,
O-acetylation on RhaIII and GlcNAc has been overlooked, and
the corresponding O-factors 9 and 10 have not been included in the
S. flexneri serotyping scheme. To fill the gap, we suggest to
further divide the existent serotypes into O-factor 9- and 10-positive
and -negative subtypes (i) by keeping the old names for the subtypes
that lack both O-factors 9 and 10 [24, 33] and for serotype 1b whose O-factor 9-negative
variant has not been found in nature, and (ii) by indication of
expression of one or both of the O-factors 9 and 10, by adding
subscript 1 or 2, respectively (e.g. 2a1 and 2a2
for subtypes characterized by the antigenic formulae II: 10 and II: 9;
10, respectively). To distinguish these subtypes, monospecific antisera
against O-factors 9 [33] and 10 [24] that have already been generated and verified
should be included into the serological diagnostic kit.

Phosphorylation with PEtN has been reported in subtypes 4av, Xv, Yv, and
Yv1 designated as “variant” subtypes by adding
letter “v” to the names of the corresponding PEtN-positive
subtypes [26-29].
Stoichiometric phosphorylation occurs at position 3 of
RhaIII in subtype 4av [26] or
RhaII in subtype Xv [27] (Fig. 1) with minor phosphorylation (~10%) on the
neighboring rhamnose residue, in subtype Xv the minor PEtN group
replacing the glucosyl group on RhaIII [27]. In subtypes Yv and Yv1, both rhamnose
residues are phosphorylated, one being completely (RhaII in
subtype Yv or RhaIII in subtype Yv1) and the
other partially modified ([28] and authors’
unpublished data). It has been demonstrated that in subtype
Yv1, bisphosphorylation occurs in the non-O-acetylated
O-units only, and the Yv1 O-polysaccharide is composed of
blocks of repeats differing in the number of PEtN groups and the
presence or absence of O-acetylation [28].

O-Acetylation on RhaIII and GlcNAc as well as phosphorylation
with PEtN has not been identified in early structural studies of S.
flexneri O-antigens, but monoclonal antibody MASF IV-1 generated
against a PEtN-positive strain of subtype 4a (now 4av) has been
included into the MASF reagents (Reagensia AB). This antibody is useful
for detection of all PEtN-positive strains, as the corresponding group
O-factor IV-1 (originally called 4X [31]) is
defined by phosphorylation with PEtN whether it occurs on
RhaII or RhaIII [27, 29]. That the same monoclonal antibody recognizes a
PEtN-associated epitope on either of the two monosaccharides is
probably due to a sharp turn of the polysaccharide chain at each
2-substituted Rha residue, which makes both RhaII and
RhaIII easily accessible to interaction with the protein and
neglects the role of the neighboring sugar residues.

The serotype 6 O-polysaccharide (2) is acidic due to the presence of
d-galacturonic acid (GalA) [10, 36]. The first monosaccharide in the serotype 6
O-unit is 2-acetamido-2-deoxy-d-galactose (GalNAc) rather than GlcNAc,
but the α1→2-linked rhamnose disaccharide at the other side
of the O-unit is shared by all S. flexneri serotypes.

Serotype 6 strains are recognized by typing antiserum VI specific to an
unidentified O-polysaccharide domain. The only modification of the
serotype 6 O-antigen that has been characterized chemically is
3/4-O-acetylation on RhaIII [10, 33]. Due to the presence of an O-polysaccharide
backbone fragment in common with non-6 serotypes of S. flexneri,
this modification confers serotype 6 with O-factor 9, which is
recognized smoothly by antiserum 9 produced against an O-factor
9-positive strain of serotype 2 [23, 33]. O-Factor 9 is present in all serotype 6 strains
tested [10, 23, 33]. As in serotype 2a, the terminal
RhaIII residue of the single O-unit in a short-chain
serotype 6 LPS is O-acetylated randomly [40].

Seven atypical serotype 6 strains collected in Bangladesh during
1985-1987 [45] and 1997-2000 [46] were recognized by monoclonal antibody MASF IV-1,
suggesting that subtype 6v carrying a PEtN-associated epitope emerged
in nature. Structural and genetic bases of the O-factor IV-1 expression
in this subtype remain to be elucidated.

Glucosylation. Three Gtr proteins (GtrA, GtrB, and type-specific
Gtr (Gtr(type)) mediate glucosylation of the O-polysaccharide backbone
(1). GtrA and GtrB are highly conserved and functionally
interchangeable between serotypes. GtrB catalyzes synthesis of
undecaprenyl phosphate-β-glucose (UndP-β-Glc) from
UDP-α-Glc, and GtrA functions as flippase allowing translocation
of the UndP-β-Glc from cytoplasm to periplasm. The third protein,
Gtr(type), is a serotype-specific glucosyltransferase (GtrI, GtrII,
GtrIV, GtrV, GtrVII (formerly GtrIc), and GtrX) responsible for the
transfer of the glucosyl group from UndP-β-Glc to a certain
position of one of the sugar residues of the growing O-polysaccharide
chain. The Gtr(type) enzymes are integral membrane proteins consisting
of 8-10 transmembrane helices with the active sites located in the
large periplasmic loops at the N- and C-termini. They have weak
similarity to other known glucosyltransferase families and are
predicted to be members of the GT-C superfamily, which utilize a
phospholipid-activated donor sugar substrate [47].

A single operon on the chromosome encoding Gtr proteins (gtr
cluster) is carried by a (cryptic) prophage acquired by lysogeny of the
bacteria with one or two from five temperate bacteriophages (SfI, SfII,
SfIV, SfV, and SfX) [14, 48].
Each prophage (or a couple of prophages in the case of bisglucosylation
in the O-unit) is integrated into the thrW tRNA gene in the same
region adjacent to the proA gene in the S. flexneri
genome. The gtr cluster is localized immediately downstream of
the phage attachment site attP, which follows the integrase
(int) and excisionase (xis) genes. All bacteriophages
have been isolated from the corresponding S. flexneri strains
and well characterized [41, 46-54].

Lysogeny with bacteriophages SfI, SfII, SfIV, SfV, and SfX converts
serotype Y to serotypes 1a, 2a, 4a, 5a, and X, respectively
(Fig. 2), whereas the potential recipient
range among other serotypes is quite different. This is single serotype
X for SfI, two serotypes (3b and 5a) for SfII, two serotypes
(2a1 and 3b) for SfX, four subtypes of serotypes 1a, VII,
and X for SfIV, and six subtypes of serotypes 1-4 for SfV of the 12
serotypes tested. The limitation in the host recognition is evidently
due to the phage immunity from a modified O-antigen, which constitutes
the receptor for the phage adsorption on the cell surface, a mechanism
by which lysogeny prevents subsequent infection of bacteria by
homologous or related phages ([52-54], Q. Sun, J. Wang, X. Luo et al., unpublished
data). Accordingly, the order of lysogeny with two phages giving rise
to serotypes carrying more than one phage-borne modification factor can
be constrained; for instance, phage SfI can infect SfX-carrying
serotype X strains giving rise to serotype 1d, but serotype 1 strains
are resistant to phage SfX [55] (Fig. 2).

Fig. 2. Conversion pathways of serotypes 1-5, 7, X, and Y
mediated by bacteriophages SfI, SfII, SfIV, SfV, SfVII, SfX, Sf6 or
Sf61b, Sf101, and plasmids pSFxv_2 or pSFyv_2. Adopted from
([24, 25, 27, 29, 52-56, 59],
Q. Sun, J. Wang, X. Luo et al., unpublished data). Antigenic formulae
are shown in parentheses (except for serotype Y, group O-factor 3,4
associated with the O-polysaccharide backbone is omitted). Asterisk
indicates gene inactivation. Dashed arrows show that the order of
serotype-converting events is unknown. Putative intermediates that have
not been found in nature are shown in dashed rectangular box.
Hypothetical bacteriophages Sf61b and SfVII have not been
isolated. The bacteriophage Sf101 origin of the oacB gene has
been demonstrated for two serotype 7a1 strains, and
mobilization of oacB into other 3/4-O-acetylation-carrying 7a
and non-7a strains suggested to result from disruption of the Sf101
prophage by IS (insertion sequence) elements followed by recombination
[25].

Inactivating mutations in the gtr locus occur in a number of
wild-type strains that carry a serotype-converting phage, resulting in
their reversion to the parental serotype (Y) or (in case of
bisglucosylation) an intermediate serotype (Fig. 2). For instance, from 35 serotype Y strains, 13
strains possess defective gene gtrII and six strains defective
gene gtrI. From 19 strains of O-factor IV-1-positive serotype Yv
and Yv1, 13 strains have mutations in either one or both
genes gtrII and gtrB, and three strains possess a
defective gtrX [29]. As a result, the same
serotype may have multiple origins, e.g. subtypes Yv and Yv1
emerged independently at least three times from serotypes Y, Xv, and 2a
by acquisition of an opt-carrying plasmid, inactivation of a
gtr gene, and both events, respectively [29] (Fig. 2).

In serotype 7 that is distinguished by the presence of the side-chain
α1→2-linked glucose disaccharide, the addition of the first
glucosyl group is mediated by the same gtr cluster within a SfI
prophage as in serotype 1 [56]. The gtrVII
gene designated originally gtrIC codes for the serotype
7-specific glucosyltransferase that mediates addition of the second
glucose residue to the first one. As the other type-specific
gtr(type) genes, gtrVII is present as part of a
three-gene gtr cluster but is located at a different place on
the chromosome adjacent to the conserved yejO locus. It is
distantly related to the other S. flexneri gtr clusters and
appears to have been acquired from outside the species, presumably via
infection by a hypothetical bacteriophage SfVII (SfIC) [56].

O-Acetylation. 2-O-Acetylation of RhaI is mediated by
an acetyltransferase, which was originally named Oac, but after
discovery of other O-antigen-modifying acetyltransferases in S.
flexneri, it was suggested to rename it OacA [22]. The receptor for OacA is the O-antigen of
serotype Y having the basal structure (1) as well as some other
serotypes (Fig. 2). OacA consists of 10
α-helical membrane-spanning regions with both the N- and
C-termini located in the cytoplasm. It bears homology to several known
and predicted acetyltransferases with most homology existing in the
N-terminal transmembrane regions [57]. In
serotypes 3a, 3b, and 4b, the oacA gene and an adjacent
integrase-encoding gene are carried by the temperate bacteriophage Sf6,
which, like the gtr locus-carrying bacteriophages, is a member
of the canonical lambdoid phage group. The Sf6 genome is integrated
into the argW tRNA gene of the host chromosome next to the
conserved yfdC gene (Fig. 3a) [58].

Serotypes 1b [59] and 7b (authors’
unpublished data) possess a variant oacA gene named
oacA1b (originally oac1b), which
shares with oacA 88-89% identity at the DNA level and 85%
identity at the protein level. Despite the rather high sequence
variation, oacA and oacA1b are functionally
interchangeable in 2-O-acetylation of RhaI.
oacA1b is located in a chromosomal region between the
conserved torT and ycmA genes, which evidently has a
phage origin but is different from the Sf6 phage genome (Fig. 3a). Whereas serotypes 3a, 3b, and 4b can be generated
by infecting with bacteriophage Sf6 strains of serotypes X, Y, and 4a,
respectively, this phage cannot convert serotype 1a into serotype 1b.
Therefore, it is likely that oacA1b has been obtained
from outside S. flexneri, probably by infection with another
bacteriophage (hypothetical Sf61b) rather than evolved by
divergence from the oacA gene.

6-O-Acetylation of GlcNAc is mediated by an oac homolog
designated oacD, which is carried by SfII bacteriophage also
responsible for 4-glucosylation of RhaI giving rise to
serotype 2 (Fig. 3b) [24]. The occurrence of an insertion sequence (IS)
upstream of oacD suggests that this gene was incorporated into
the SfII genome by an insertion event. The functional oacD gene
also is present in strains of several non-2 serotypes (3a1,
X1, Y1, Y2, and Yv1) that
carry a cryptic SfII prophage with a dysfunctional gtr locus for
the type II glucosylation. Therefore, the OacD-mediated 6-O-acetylation
of GlcNAc does not depend on the functional gtr locus.

3/4-O-Acetylation of RhaIII that occurs in subtypes
1a1, 1b, 2a2, 5a1, 71,
Y1, 6, and Y2 [22, 25] is mediated by another Oac homolog called OacB.
This modification was unaffected by transformation of 2a2
and Y1 strains with either the gtrABX locus for
3-O-glucosylation (authors’ unpublished data) or the optII
gene for 3-O-phosphorylation [60] of
RhaIII. In contrast, transformation of 2b and X strains with
oacB from a 2a2 strain resulted in their conversion
into serotypes 2a and Y, respectively, due to replacement of
3-O-glucosylation with 3/4-O-acetylation on RhaIII [22]. The mechanism that makes the O-acetylation the
preferable modification on RhaIII remains to be
elucidated.

Two alternative locations of the oacB gene on the bacterial
chromosome have been reported. In some subtype 7a1 strains,
oacB is carried by Sf101 prophage integrated in the sbcB
gene next to yeeD [22] (Fig. 3b). In several different subtype 7a1
strains [25] and 3/4-O-acetylation-carrying
strains of other serotypes [22], oacB maps
upstream of the adrA gene in the same proA-adrA
region on the chromosome, in which the gtr-carrying prophages
are integrated. It is located downstream of an integrase-encoding gene
(int), and the int-oacB locus is flanked by IS
elements giving rise to a transposon-like structure [22]. In serotype 2a strains examined, this structure
is located immediately downstream of the SfII prophage genome
(Fig. 3b).

In serotype 6, yet another oac homolog, oacC, common for
all strains of this serotype, is responsible for the 3/4-O-acetylation
of RhaIII (Fig. 3c) [23]. It maps in a phage-like structure localized in
yet another place on the chromosome (Fig. 3c).
OacB and OacC have high sequence homology (72% identity) and
interchangeable function in mediating the 3/4-O-acetylation of
RhaIII. This is not surprising as the O-polysaccharides of
all S. flexneri serotypes share a
→2)-α-l-RhapIII-(1→2)-α-l-RhapII-(1→
disaccharide fragment, which evidently serves as the acceptor substrate
for both OacB and OacC. The three known rhamnose-modifying
acyltransferases OacA-OacC present higher homology in the regions
conserved among the inner membrane trans-acylase family proteins
[23]; particularly, conserved are amino acid
residues R73 and R76, which are known to be critical for Oac
functioning [57]. The divergent oac genes
might have been gained from different bacterial species in independent
events.

Phosphorylation with PEtN. A polymorphic opt gene
(originally called lpt-O) encoding for PEtN transferases is
responsible for adding a PEtN group to RhaII or/and
RhaIII in subtypes 4av, Xv, Yv, and Yv1 [27, 28]. The Opt proteins have
been predicted to belong to the sulfatase superfamily and to contain a
sulfatase domain on the carboxyl terminus, which putatively is involved
in catalyzing the transfer of PEtN to a sugar residue. Two functionally
interchangeable opt alleles, optII and optIII, are
borne by double-stranded circular plasmids 6850 bp in length called
pSFxv_2 and pSFyv_2, respectively (Fig. 4).
OptII and OptIII preferentially mediate phosphorylation of
RhaII and RhaIII [27-29], and, accordingly, optII and optIII
are present in subtypes Xv and 4av, respectively. An explanation for
the evolution of this gene may be selection pressure as in serotype 4a,
RhaIII is not occupied, and the OptIII can easily mediate
the addition of PEtN onto it. In contrast, in serotype X,
RhaIII carries a glucosyl group, and the
OptIII-mediated phosphorylation may not effectively compete for
RhaIII with type 7,8 glucosylation, whereas OptII can
smoothly modify RhaII. In subtypes Yv and Yv1,
the opt form depends on the strain origin: this is optII
in Yv strains derived from serotype Y or Xv or optIII in
serotype 2-derived Yv1 strains (Fig. 2) [29]. Accordingly,
RhaII and RhaIII are predominantly phosphorylated
in subtypes Yv and Yv1, respectively ([28] and authors’ unpublished data).

It has been demonstrated that plasmids pSFxv_2 [60]
and pSFyv_2 (authors’ unpublished data) can be transferred into,
and stably maintained in, strains of other S. flexneri serotypes
(1 to 6) giving rise to unnatural O-factor IV-1-positive serovariants.
Upon the transformation, the initial serospecificity is either retained
or lost, or manifestation of an initial epitope(s) may be weakened.
Phosphorylation with PEtN may interfere with other modifications on the
O-antigen not only on the same monosaccharide but also on different
sugar residues; for instance, 3-O-phosphorylation on RhaII
is incompatible with 4-O-glucosylation on RhaI [60]. This phenomenon seems to account for the absence
of the PEtN-carrying variant of 2a (subtype 2av) in nature, while its
non-phosphorylated form is highly prevalent among clinical isolates. It
also shows that in the emergence of subtype Yv1 from subtype
2a1, inactivation of gtrII or gtrB and the
resultant loss of type II glucosylation occurred first and pSFyv_2
plasmid was gained subsequently (Fig. 2).

DISCUSSION

Known modifications of the O-antigens of S. flexneri involve
glucosylation, O-acetylation, or/and phosphorylation with PEtN at
various monosaccharides in the O-unit. None is unique on its own, but
their most diverse combinations provide an unusually high diversity of
the O-antigen forms. By now, about 30 structural variants of the
O-antigens of S. flexneri with the same basal structure have
been identified, which exceed significantly their number in any other
bacteria.

Glucosylation has been known to take place at the periplasmic side of
the inner membrane together with the O-unit polymerization [14]. The data on the O-acetylation summarized earlier
[14] are contradictory, and it remains unknown
where phosphorylation of the O-antigen takes place. Some modifications
on different monosaccharide residues, e.g. 4-O-glucosylation on
RhaI and 3-O-phosphorylation on RhaII, are
incompatible with each other. The modification pattern of the first
O-unit that is linked to the LPS core may be different; particularly,
glucosylation only happens to at least the second O-unit from the
growing end, the first one being unaltered [40].
This finding and the fact that the same Wzy polymerase catalyzes
synthesis of structurally different O-polysaccharides indicate complex
interactions of the enzymes involved in the O-antigen polymerization
and modifications, which remain to be elucidated.

The diverse O-polysaccharide modifications are encoded by a number of
genes outside the O-antigen clusters between galF and
gnd, and their roles have been elucidated by serological
analysis of the wild-type and non-polar mutants and structure
determination of their isolated O-polysaccharides. Multiple genetic
mechanisms have been recognized to underlie the S. flexneri
O-antigen modifications and the resultant serotype conversions.

Most common is lysogeny by bacteriophages that encode
glucosyltransferases and/or acetyltransferases. One of the
bacteriophages, SfII, includes both gtr gene cluster for
4-O-glucosylation of RhaI and oacD gene for
6-O-acetylation of GlcNAc [24]. This is a unique
situation in S. flexneri that one serotype-converting phage
carries two genetic factors involved in different types of O-antigen
modifications.

Another way of O-antigen modification factor mobilization is a
combination of the oacB gene for 3/4-O-acetylation of
RhaIII with several IS elements giving rise to a
transposon-like structure [22], which could evolve
as a result of disruption of oacB-carrying Sf101 prophage by IS
elements [25].

Most gtr-containing prophages and the oacB-carrying locus
map in the same region on the chromosome upstream of the adrA
gene, which seems to be a conserved insertion site for mobile genetic
elements. On the other hand, the gtr locus for addition of the
second glucose residue in serotype 7 as well as oacB in some
subtype 7a1 strains and genes for all other
acetyltransferases are parts of bacteriophage genomes or phage-like
structures integrated at different places on the chromosome. The
polymorphic opt gene that encodes for PEtN-transferases
responsible for phosphorylation of RhaIII or/and
RhaII, is carried on 6.85-kb plasmids, which have high
dissemination potential among S. flexneri serotypes ([60] and authors’ unpublished data).

Therefore, the primary genetic mechanism of diversification of S.
flexneri non-serotype 6 O-antigen structures is acquisition of
various transferable genetic factors, including prophages and plasmids,
which can easily spread among different strains. Inactivation of a gene
involved in an O-antigen modification, such as a gtr gene,
oac, or opt, contributes to further conversion of
serotypes. Because of the involvement of multiple O-antigen
modification factors, the same serotype may emerge multiple times not
only by the same [61] but also by different ways,
including both gaining and losing one or several factors (Fig. 2).

Recent identification of PEtN phosphorylation and O-acetylation at new
sites has refined the notion of the antigenic heterogeneity of S.
flexneri (table and Fig. 2). It has
become clear that the diversity of the O-antigenic forms in these
bacteria was underestimated in the past. For their separate detection,
it was proposed to apply specific antisera obtained by absorption of
immune sera against wild-type strains with the corresponding isogenic
mutants or vice versa [23, 24, 27, 28,
33, 62, 63], as well as molecular approaches using as targets
the specific genes responsible for the O-antigen modifications [22-24, 27,
28, 61]. Serological and
molecular screening showed that most newly found O-antigen forms occur
rather frequently. The data show the expediency of an extension of the
existing S. flexneri serotyping scheme by recognition of the
representative strains of the new variants as distinctive subtypes.

Human immune response to S. flexneri infection is serotype
specific with protection against subsequent infection by the same
serotype only. As acquisition of multiple drug resistance, the
appearance of new surface epitopes due to O-antigen modifications would
be expected to offer a significant advantage to the pathogen and to
promote its spread in human populations. For instance, serotype Xv
distinguished by PEtN phosphorylation of RhaII appeared
initially in one province in China in 2001 and rapidly expanded to most
provinces, surpassing 2a as the predominant serotype [62]. It has been demonstrated that glucosylation on
RhaI, RhaII, and GlcNAc confers a specific
advantage on S. flexneri [16], and it may
be suggested that 3/4-O-acetylation on RhaIII is somehow
beneficial to the bacteria too, as it occurs in >95% strains of
serotypes 1a, 1b, and 2a, which are predominant in developing
countries.

Therefore, the data presented in this review, including elucidation of
the finer details of the O-antigen modifications and the underlying
genetic mechanisms, shed light, and provide avenues for further
studies, on the role of the O-antigen variations in the antigenicity,
pathogenicity, and epidemicity of S. flexneri. They also have
profound implications in development of improved diagnostic methods and
efficient shigellosis vaccines targeting newly discovered genes and
epitopes.

We acknowledge our colleagues who have significantly contributed to the
advancement of this field.

This work was supported by the Russian Science Foundation (Grant No.
14-14-01042 for Y. A. K.). Q. S. and J. X. were supported by the
National Natural Science Foundation of China (No. 81271788, 81290340
and 81290345) and the National Key Program for Infectious Diseases of
China (2013ZX10004221, 2013ZX10004216-001-002).